ARFs (ADP ribosylation factors) are members of a large family of GDP/GTP switch proteins that are key elements in the control of bi-directional membrane traffic between the endoplasmic reticulum, the Golgi, endosomes, and the plasma membrane. Regulation of membrane traffic is very important to the maintenance of proper lipid and protein composition at the cell surface as well as in intracellular organelles. It is also integral to the transmission of various intra- and inter- cellular signals. In general, ARFs function by interacting with membranes through an N-myristoyl fatty acid chain, an N-terminal amphipathic helix, and specific predicted binding sites for lipids and other proteins. This process is correlated with GDP to GTP exchange and hydrolysis, under the influence of various protein effectors, exchange factors, and GTPase activators. The process results in recruitment of various components to nascent areas of transport, membrane deformation, and fission of a vesicle carrier. However, the way in which the biochemical tranformations of ARF are coordinated with carrier formation, and the structural aspects of membrane association and protein-protein interaction are not well understood. During the past funding period, solution NMR methodology for the study of membrane-associated ARF structures has been devised, and structures of both GDP and GTP forms of the full-length, myristoylated yeast ARF1 protein have been produced. Application of this methodology to an understanding of the the interaction of ARF with other proteins and lipids is now proposed. More specifically: 1) Interactions of ARF with a series of membrane mimetics will be examined to establish correlations between surface curvature, binding affinity, and structural alterations. 2) Binding of ARF to phosphoinositides incorporated in lipid membranes will be characterized and the structures of ARF-phosphoinositide complexes will be determined. 3) The interaction of an ARF effector protein (FAPP1) with both phosphoinositides and ARF will be biochemically and structurally characterized. 4) Potential targets for structural characterization of a second ARF-interacting protein will be investigated. These include a guanine nucleotide exchange protein (RafF) from the causative organism of Legionnaire's Disease and a GTPase activating protein (ArfGAP1). The most suitable of these systems will be selected for more complete structural characterization. The results will provide an improved picture of ARF's role in vesicle carrier generation, and in the process, novel NMR approaches to the structural characterization of membrane-protein complexes will be developed and exported to the scientific community.